Abstract Neutrophils are the most abundant leukocytes in humans and essential for innate immunity and inflammation, including cardiovascular inflammatory diseases, such as ischemia-reperfusion injury (IRI), post- myocardial infarction inflammation and atherosclerosis. Arrest is a key step in neutrophil recruitment from blood to inflamed tissues. Neutrophils arrest on activated endothelium under flow using the beta2 integrins. My previous work confirmed the known pathway of beta2 integrin activation (extension E followed by headpiece- opening H; E-H- to E+H- to E+H+) and discovered a new pathway where the headpiece opens before the integrin extends during arrest of primary human neutrophils (E-H- to E-H+ to E+H+). The newly identified bent-open (E- H+) beta2 integrin binds ligands (ICAMs) expressed on neutrophils in cis. I showed that this auto-inhibition limits neutrophil adhesion in vitro and in vivo. The proposed work will, for the first time in this field, interrogate beta2 integrin activation by super-resolution microscopy. I refined the preliminary data by molecular modeling and achieved single molecule resolution. I found that E-H+ integrins are not randomly oriented, but show a molecular pattern consistent with a ?Face-to-Face? orientation. In specific aim 1, I will test the hypothesis that this ?Face-to- Face? pattern is caused by pairwise in-cis interactions of E-H+ integrins binding to ICAM dimers. If so, function- blocking ICAM antibodies should disrupt this ?Face-to-Face? pattern. Non-blocking ICAM antibody will be used to test whether ICAMs are co-localized with E-H+ beta2 integrins as expected. In specific aim 2, I will screen small molecule allosteric inhibitors that keep beta2 integrins in the auto-inhibited E-H+ conformation. In my preliminary experiments, I already developed flow-cytometry-based high-throughput screening of integrin activation (E+ and H+). I will test compounds in three libraries to find candidates. I propose to confirm the efficacy of successful candidate molecules in primary neutrophils using flow cytometry and established microfluidic adhesion assays. Specific aim 3 is to directly test the physiologic significance of E-H+ integrins. I will test the hypothesis that auto-inhibition of E-H+ beta2 integrins protects cardiomyocytes from IRI. I will use mice transplanted with ICAM-1 and ICAM-2 double knockout bone marrow, which I have previously shown to eliminate the auto-inhibition of beta2 integrin on neutrophils. I expect these chimeric mice to show more severe myocardial IRI and tissue loss. Successful inhibitors from aim 2 will be tested in this IRI model in vivo. After completion of these experiments, we will know the molecular details of beta2 integrin activation during arrest of primary human neutrophils (aim 1), find candidate small molecule inhibitors that stabilize the E-H+ beta2 integrin conformation (aim 2), and know the in vivo relevance of E-H+ beta2 integrins (aim 3). The candidate inhibitors represent lead compounds for drug development aimed at for preventing and treating inflammatory diseases, such as IRI and chronic vascular inflammation.